Posts Tagged ‘agarose’

I wanted to find a cheap way to make electrophoresis buffer from easily accessible ingredients and there were two pieces of knowledge that led me to this experiment. First I had read about some labs using sodium borate (SB) buffer because it gave great results. Secondly, I knew that borate and boric acid were easy to find at stores in the forms of roach poison (boric acid) and borax (sodium tetraborate).

Originally I had intended to compare a large number of buffers; TAE, TBE, SB (molecular biology grade), and SB (home grade). Due to my own personal shortages of lab-grade materials I had reduce down to a comparison between TBE, SB (home grade), and SB with EDTA (home grade).

All gels were 1% agarose. Gel images were made with Foto/Phoresis I transilluminator and gel images were recorded with an iPhone 3GS camera.

Tris-Borate-EDTA (TBE)

Purchased from the “Online Science Mall” as a 5X concentrate. Without a doubt this worked better than the buffers I cobbled together.

SB Buffer (home-grade)

SB buffer made by adding 1.91g of Sodium borate decahydrate (Borax by 20-mule team) to ~800mL distilled H2O (Target), pH was adjusted with 0.4M Boric Acid (Roach Away by Enoz) and then diluted to final volume with more distilled water.

SB Buffer with EDTA (home-grade)

A 600 mL aliquot of the SB buffer, made previously, had 1.2 mL of 0.5 M EDTA (molecular grade) added to reach 2mM final concentration.

I decided to test the SB buffer with EDTA to see if there were any nuclease related problems occurring. 2/3rds of the way through the project it occurred to me I was using Target brand distilled water instead of nuclease free water when mixing the 2-log DNA. It just simply slipped my mind. So this third gel buffer test was done with EDTA and nuclease free water to see if the results would be drastically different or not and they were not. I believe any differences in the images is due to my lack of a proper gel documenting system.

Given the choice between the three buffers, TBE is the superior. I do not mean to say that one could not use the SB home grade buffers and get usable results, but rather they just are not as good. Being that store bought TBE and TAE are not crazy-expensive and buffers can be reused a couple of times, there is not a lot of impetus to use home-grade SB buffers.

My results for the SB buffer tests do not even come close to mirroring the results from the Brody paper I linked earlier, I suspect that my preparation or formulation of SB may be flawed.

While unfortunate that I couldn’t test more buffers, I was happy to figure out that TBE will likely work for my PCR projects in the future and if I am ever mismanage my supply of TBE I now know I can make up some SB buffer to get by.

I will run the test again on a better gel rig (that I dislike using because it uses 3x as much agarose and gel stain as 1 mini gel I used below).

All gels were run at 100V for 1 hour.

SB Home Grade (left). SB Molecular Grade (center). 1 TBE (right).

1x TAE (left). 1x TBE (right).

You can see that the TAE buffer led to gel deformation whereas TBE did not have that problem.

From left to right. TAE, SB Home-grade, SB Molecular-grade, TBE.

The two SB buffers were noticeably cooler than the TBE and TAE buffers (27C for SB and 33C for TBE and TAE) and the DNA migrated slower.

I think most of the smearing is due to the high voltage used. The crappy 2-log ladder (that I have had issues with before) also doesn’t help.

Another problem is my power supply doesn’t handle four simultaneous gels very well, the mA jump up super high in order to generate the needed voltage. I will be running them individuals on the next test.

Gel electrophoresis is a core technique used in molecular biology laboratories. The gels used for electrophoresis are almost always agarose which is a polysaccharide purified from agar-agar. Agar-agar is obtained from red algae and contains a variety of impurities along with the agarose. Agar-agar is often used as a substitute for gelatin in vegetarian dishes and is used to make some cool culinary creations. Pure agarose is obtained by separating it from agar-agar. I have heard from variety of sources that agar-agar could be used in place of agarose for gel electrophoresis of DNA. Given that agarose costs around $1.00 per gram and agar-agar costs around $0.05 per gram I thought it was worthwhile to check the efficacy of agar-agar. My results show that agar-agar is not an acceptable substitute for agarose (see the image above).

For more details and thoughts read the rest of this post.

Cleaning the Agar-agar

The night before I ran the experiment I decided that it would be nice if I tried to “clean” the agar-agar of some impurities and run this as an additional treatment group. Since I did this last minute I did not completely think my plan of attack through and actually made the agar-agar worse. I used 50% isopropanol and distilled water to rinse the agar-agar through a coffee filter.

After running the experiment and seeing my results I did the research I should have done prior to the experiment. A quick google search showed me a technique for purifying agarose from agar-agar using propylene glycol. Click here for the site in question.

Failed Purification Protocol:

Weighed out 11.7g agar-agar into a coffee filter (2 thick).

Saturated agar with distilled H2O (dH2O).

Poured 100mL of dH2O through the slurry and allowed it to drain.

Stirred slurry with a spatula.

Pourd 100mL of dH2O through the slurry and allowed it to drain.

Stirred slurry with a spatula.

Poured 100mL of 50% isoproanol into the slurry.

Stirred slurry with a spatula.

Once the majority of fluid drained out I placed the agar-agar and coffee filter into an oven at 90C for 5 hours to dry.

Experiment Parameters

1.2% gel

TAE Buffer

2.5 hours

70 volts

Biotium GelRed Stain (used as a precast additive)

Promega 1kb DNA Ladder (left lane)

NEB 2-Log DNA Ladder (right lane)

Pictures and Comments

Agarose gel atop the UV transilluminator.

I had minor issues taking pictures of the gels because too much light was reaching the camera lens so I made a aluminum foil frame to block out extra light.

All 3 gels deformed on the end closest to the (-) electrode and I am not sure why. I suspected temperature at first but it subjectively seemed constant throughout the gel rig. Next I thought it was the gels positioning, however when checked I found each end to be equally distant from both electrodes.

Compare the picture above to the reference gel below (in black and white). This reference gel was the first gel I ran with my gel rig and it ran for 80min at 70V using the same TAE buffer used in this experiment and there was no deformation of the gels wells. The two differences between the reference gel and the newest gels were time and staining compound (2.5 hours vs. 80min and ethidium bromide vs. GelRed). If this problem persist I will have to run a bunch of gels to figure this out.

See blurb above for information about this reference gel.

All 3 experimental gels on the UV transilluminator. From left to right; agar-agar, cleaned agar-agar, and agarose.

Conclusions

Based on my results I do not recommend purchasing agar-agar for gel electrophoresis. The only exception I would make is if the intent is to use propylene glycol to separate the agarose from the agar-agar (click here for agarose separation with propylene glycol). Keep in mind that there could be nuclease and cation contamination in the propylene glycol purified agarose.

While agarose is a lot more expensive than agar-agar, it is not expensive relative to everything else needed for conducting molecular biology at home. For the sake of simplicity, reliability, and time I intend on using only agarose going forward.

What is next

My next post will compare running buffers between gels – specifically molecular biology grade Tris-Acetate-EDTA (TAE buffer), Molecular Bio grade sodium borate buffer (SB Buffer), and sodium borate buffer made from borax and cockroach poison (boric acid). If there is any simple household substances you want me to try as a running buffer, let me know. I have a lot of school and work projects going on so this upcoming post may be a few weeks out.

Gel boxes are ridiculously expensive, so much so that I was able to buy a used electrophoresis machine off eBay for less than the cost of a gel box!

My first attempts in building a gel box involved scoring and sawing 1/4″ plastic boards in an attempt to cut square pieces for a gel box. This method was too crude and I could not get a straight line. The only positive thing from this attempt was I found that PVC cement was able to join plastic pieces together to make a strong water-tight seal.

While brainstorming I reckoned that LEGO’s would be perfect for a gel box. After googling for a bit I found only one reference to LEGO’s and electrophoresis and it was at the journal of BioTechniques (link here). It was frustrating to discovery that no digital copy of the article exists and so I decided I would fumble around in the dark on my own.

Casting a gel with LEGOs

The image above shows my prototype gel box and gel mold (with molten agarose). The box itself is just a frame. The gel mold is square so that the mold can be rotated once the agarose cools to make for easy gel casting. The bottom of the mold is covered in those smooth LEGO planks without rivits (I suspect it may have worked even with the rivits, but the acetone treatment would be more difficult).

One of the big design challenges with using LEGO’s is waterproofing the construct. LEGO’s are made from ABS plastic and from all of the reading about 3D printers I have done, I knew that acetone melts and melds ABS plastic together. After constructing my gel mold I dipped a Kim-Wipe (lint free paper towel) into some acetone and rubbed it all over the sides and surfaces of the LEGO mold. Immediately the colors began to smear and the cracks and grooves filled in. Within minutes the whole thing was dry and ready for casting a gel.

Nearly all of the agarose stayed inside the mold (I added 40mL of 2% agarose) and the mold and gel both were removed and rotated nicely!

I ran into one issue. The acetone was not completely dry and it seeped up from the base plate of LEGO’s and made a weird tiny plastic ridge (which I later removed with more acetone). I only gave the LEGO’s about 5 minutes to dry so I am not surprised. But what was surprising was the patriotic shape theplastic made in my gel!

Flag? Gel?

I am definitely going to pursue the LEGO gel box further and will post more if I have success.